This invention relates generally to analytical chemistry, and more specifically to quality assessment of polymer synthesis reactions, such as oligonucleotide synthetic reactions.
Oligonucleotides are among the most ubiquitous reagents used in biotechnology laboratories engaged in research, diagnostics and therapeutics. The high demand for oligonucleotides derives from their exquisite specificity for complementary nucleotide sequences in DNA or RNA molecules obtained from biological samples. This specificity allows oligonucleotides to be used as probes to identify a unique sequence present in less than part-per-billion abundance, for example, in order to provide a diagnosis for an individual at risk for a particular disease. Furthermore, this specificity also forms the basis for use of oligonucleotides as reagents for synthesizing molecules of DNA or RNA having a particular nucleotide sequence of interest. For example, a gene sequence associated with a particular disease can be cloned by hybridizing one or more oligonucleotides to the gene sequence and performing an amplification reaction to make multiple copies of the sequence. The cloned gene sequence can subsequently be utilized for research into the disease or can even be used for therapeutic treatment of individuals afflicted with the disease.
A variety of synthetic methods useful for producing oligonucleotides at various production rates are known in the art. Synthetic throughput can range from a production rate of just a few oligonucleotides per day, for example, by a small research lab making its own oligonucleotide reagents, to over 17 million oligonucleotides per year, for example, by a manufacturing facility providing commercial oligonucleotides to a worldwide market. Typical oligonucleotide synthetic methods are relatively robust being capable of handling oligonucleotides of varying length from just a few nucleotides per molecule to over 100 nucleotides per molecule. Furthermore, the methods are capable of producing oligonucleotides having a myriad number of different sequences, the complexity of which is illustrated by the fact that the number of different decamers (molecules having 10 nucleotides) that can be made using just the 4 common DNA nucleotides (A, T, C and G) is 410=1,048,576.
Although currently available synthetic methods allow large numbers of oligonucleotides to be synthesized in a short period of time, the time and resources currently required for performing quality assessment typically impose a bottleneck on the overall rate of oligonucleotide production. Traditional methods of assessing the purity and sequence constitution of oligonucleotides include analytical techniques such as mass spectroscopy, capillary electrophoresis, UV absorbance spectroscopy and slab gel electrophoresis, which typically require manual implementation and/or evaluation by trained technicians. The desire to produce oligonucleotides at a rapid rate and at low cost to satisfy the ever increasing demand while staying competitive in the marketplace can increase pressure on manufacturers to minimize use of such methods to the point that quality assessment is based on a subset of reactions that is too small to be sufficiently representative of the manufacturers output. In some cases the pressure may lead to avoidance of empirical quality assessment, relying instead on theoretical or supposed robustness of the manufacturing process. However, such shortcuts in quality assessment place the burden on the end user to either evaluate the reaction product independently or risk reagent failure in an important experiment or procedure. The risk of reagent failure can be quite large and, in some cases, unacceptable.
Thus, there exists a need for efficient methods to assess quality of oligonucleotide synthetic reactions including, for example, methods that can be readily automated. The present invention satisfies these needs and provides other advantages as well.